Ear tissue scaffold implant for auricular tissue reconstruction

ABSTRACT

Ear implants for auricular tissue reconstruction in a patient are provided. The ear implant may be a tissue scaffold multicomponent assembly for reconstruction of auricular tissue. Thus, the assembly may include both a first and a second tissue scaffold component. Each comprises a biocompatible polymeric material having a plurality of open pores configured to support cell growth. The first tissue scaffold component defines a central void region and at least a portion of an outer ear framework of the patient after implantation. The second tissue scaffold component defines a base portion. After implantation into the patient, the second tissue scaffold component seats within the central void region of the first tissue scaffold component, so that the second tissue scaffold component is secured to the first tissue scaffold component. Methods for reconstructing auricular tissue in a patient using such ear implant tissue scaffolds are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/608,716, filed Oct. 25, 2019, which is a U.S. National PhaseApplication under 35 U.S.C. 371 of International Application No.PCT/US2018/029575, filed on Apr. 26, 2018, which claims the benefit ofU.S. Provisional Application No. 62/490,312, filed on Apr. 26, 2017. Theentire disclosures of the above applications are incorporated herein byreference.

FIELD

The present disclosure relates to ear implant devices that serve astissue scaffolds for tissue reconstruction and growth and methods formaking and using the same.

BACKGROUND

This section provides background information related to the presentdisclosure which is not necessarily prior art.

Ear malformations are among the most difficult to reconstruct due totheir complex geometry. Where an unsalvageable auricle arises due totrauma, oncologic resection, microtia, anotia, and the like, auriculartissue reconstruction poses one of the most technically challengingsurgical procedures for reconstructive surgery. Children with facialdeformities, including microtia and anotia, develop self-awareness atapproximately 4 years of age and are targeted for developmentallyharmful teasing and bullying. Many patients with congenital eardeformities suffer from visual impairment as well. The external ear iscritical to provide support for prescription glasses, and thus a childmay be further deprived of optimal vision. Patients with microtiafrequently find that their glasses are poorly supported, rendering themuseless to aid in vision. This poses the potential for deprivation ofmultiple senses and further isolation of the child. Failure to restoresize, shape, and function to the ear can result in catastrophicpsychosocial harm.

Currently available treatment options to treat ear defects includeautogenous cartilage reconstruction, use of prostheses, includingsynthetic alloplastic prosthetics, or observation. Autogenous cartilagereconstruction involves carving an autogenous rib framework asfoundational support for overlying soft tissue. In this procedure, ahighly skilled surgeon carves an auricular cartilage framework forimplantation. These techniques demand the highest level of surgical andartistic ability as they rely on freehand carving of autologouscartilage, much like carving a sculpture. Even in the best of hands,outcomes can be inconsistent and undesirable. This technique can furthersuffer from other limitations, including introducing multiple surgicalsites to the patient (including a site to remove rib tissue, along withthe implantation site), as well as being a highly complex surgery withonly a limited number of surgeons available who can perform it.

The current commercially available synthetic implants may be formed of arigid synthetic polymeric material, such as high density porouspolyethylene, like the MEDPOR™ ear implant commercially available fromStryker Corp. Benefits of using a commercially available syntheticimplant include avoiding donor site morbidity from rib harvesting andlower variability with the framework appearance, in that the technicallydemanding hand carving is not required for the framework. However,commercially available implants do not have customizable framework forpatient-specific anatomy. Only a single available ear implant device isavailable to meet the needs of the wide range of pediatric and adultpatients needing reconstruction. Furthermore, rates of fracture,exposure, extrusion, and infection of porous polyethylene are believedto be unacceptably high. Implants formed from rigid, synthetic material,like high density polyethylene in conventional prosthesis devicedesigns, have a greater incidence of framework extrusion, dehiscence,and soft tissue necrosis than autogenous cartilage reconstruction. Thesecomplications frequently require subsequent operations and anestheticexposures with associated risks to address these complications. Finally,synthetic implants, like the MEDPOR™ implant device, do not have thecapacity for growth, nor do they serve as a platform for cell seeding ortissue ingrowth.

It would be desirable to provide an ear implant that serves as a tissuescaffold that can be used on a variety of patients, ranging from thevery young to older adult patients, allowing for increased projectionand scaffold expansion, along with the structural stability and abilityto withstand contraction and distortion and experiencing fewercomplications after implantation. It would also be desirable to provideprocedures that use low cost, common surgical tools that can be employedby most surgeons and even implemented in a medical mission setting.

SUMMARY

This section provides a general summary of the disclosure, and is not acomprehensive disclosure of its full scope or all of its features.

In various aspects, the present disclosure contemplates an ear implantfor auricular tissue reconstruction in a patient. In certain aspects, animplant assembly for reconstruction of auricular tissue in a patient isprovided that comprises a first tissue scaffold component and a secondtissue scaffold component. The first tissue scaffold component comprisesa first biocompatible polymeric material having a plurality of openpores configured to support cell growth. The first tissue scaffoldcomponent defines a central void region and at least a portion of anouter ear framework of the patient after implantation. The second tissuescaffold component comprises a second biocompatible polymeric materialhaving a plurality of open pores configured to support cell growth. Thesecond tissue scaffold component defines a base portion afterimplantation into the patient. The second tissue scaffold componentseats within the central void region of the first tissue scaffoldcomponent, so that the second tissue scaffold component is secured tothe first tissue scaffold component.

In other aspects, the present disclosure contemplates a method forreconstructing auricular tissue in a patient. The method comprisesimplanting a first tissue scaffold component in an ear region of thepatient. The first tissue scaffold component comprises a firstbiocompatible polymeric material having a plurality of open pores thatsupport cell growth after the implanting. The first tissue scaffoldcomponent defines a central void region configured to receive a secondtissue scaffold component and at least a portion of an outer earframework of the patient. The second tissue scaffold component seatswithin the central void region of the first tissue scaffold componentand defines a base portion of an implant assembly comprising the firsttissue scaffold component and the second tissue scaffold component.

Further areas of applicability will become apparent from the descriptionprovided herein. The description and specific examples in this summaryare intended for purposes of illustration only and are not intended tolimit the scope of the present disclosure.

DRAWINGS

The drawings described herein are for illustrative purposes only ofselected embodiments and not all possible implementations, and are notintended to limit the scope of the present disclosure.

FIGS. 1A-1F show an image-based design approach using medical images orother data specific to a patient to customize the size of an ear implantdevice prepared in accordance with certain aspects of the presentdisclosure.

FIGS. 2A-2B show computer-Aided Design and Three-Dimensional PrintingProcess for Production of Porous Bioresorbable Tissue EngineeringScaffolds. FIG. 2A show a rendering of a stereolithography (.STL) filefor a cylindrical tissue engineering scaffold with 2.7 mm spherical poreinternal microarchitecture. FIG. 2B shows a tissue engineering scaffoldmanufactured via selective laser sintering three-dimensional printingtechnique after cell seeding in a hyaluronic acid/collagen hydrogel andimplantation into four randomized quadrants in a subcutaneous pocket onthe dorsum of an athymic rat.

FIG. 3 shows a multicomponent assembly ear implant tissue scaffolddevice prepared in accordance with certain aspects of the presentdisclosure compared to a conventional commercially available highdensity polyethylene ear implant device.

FIGS. 4A-4D show a multicomponent assembly ear implant tissue scaffolddevice prepared in accordance with certain aspects of the presentdisclosure, where the figures on the left side show thecomputer-assisted-design models and the pictures on the right side showthe laser-sintered polymer scaffold structure formed based on themodels. FIG. 4A shows a top view of the first tissue scaffold component.FIG. 4B shows a perspective view of the second tissue scaffoldcomponent. FIG. 4C shows a top view an assembly of the first tissuescaffold component secured to the second tissue scaffold component. FIG.4D shows a side view the assembly of the first tissue scaffold componentsecured to the second tissue scaffold component.

FIGS. 5A-5C show another multicomponent assembly ear implant tissuescaffold device prepared in accordance with certain other aspects of thepresent disclosure. FIG. 5A shows a first tissue scaffold componenthaving an expandable opening. FIG. 5B shows the multicomponent assemblyear implant tissue scaffold device including a second tissue scaffoldcomponent seated with a central void region of the first tissue scaffoldcomponent to facilitate outward expansion of the first tissue scaffoldcomponent after implantation. FIG. 5C shows a direction of expansion ofthe first tissue scaffold component near the expandable opening.

FIG. 6 shows a perspective top view of a first tissue scaffold componentof a multicomponent assembly ear implant tissue scaffold device preparedin accordance with certain aspects of the present disclosure having aninternal open channel disposed therein.

FIG. 7 shows a perspective lower view of the first tissue scaffoldcomponent in FIG. 6 showing a drain port opening.

FIGS. 8A-8D are photographs showing auricular constructs with twomicropore architectures, random and spherical. FIG. 8A shows a randomlydistributed pore architecture in an ear implant tissue scaffold deviceprepared in accordance with certain aspects of the present disclosure.FIG. 8B shows a regularly distributed pattern of spherical pores in anear implant tissue scaffold device prepared in accordance with certainaspects of the present disclosure. FIG. 8C shows an ear implant tissuescaffold device placed in a custom-designed mold that prevents leakageof a cell-collagen solution prior to gelation. FIG. 8D shows the earimplant tissue scaffold device after gelation of the cell-collagensolution on the surface.

FIGS. 9A-9C show photographs of subcutaneous implantation of ear implanttissue scaffold devices that result in excellent external appearance ofboth anterior and posterior auricular surfaces. FIGS. 9A-9B show tissuescaffold landmarks of the ear structure, including helix, antihelix,conchal bowl, tragus, antitragus, and intertragal incisor are readilyevident after subcutaneous implantation. FIG. 9C shows projection isapproximately 25-30° off horizontal plane of the animal dorsum.

FIGS. 10A-10B show histologic analysis displays of Safranin O stainingto show cellular growth for the spherical pore scaffolds in comparisonto random pore scaffolds. FIG. 10A shows Safranin O staining for an earimplant tissue scaffold device having random pore architecture scaffold.FIG. 10B shows Safranin O staining for an ear implant tissue scaffolddevice having a spherical pore structure.

FIG. 11 shows histologic and immunohistochemical results of co-cultureexperimental groups at differing ratios of adipose-derived stemcells-to-chondrocytes after 4 weeks of in vitro followed by 4 weeks ofin vivo culture. ASC=Adipose-derived stem cells. PC=Primarychondrocytes.

FIG. 12 shows a ten times magnification of Safrinin O staining of 5:1ADSC-to-chondrocyte experimental groups after 4 weeks of in vivo growth.A white asterisk denotes well defined lacuna around chondrocytes withincartilage matrix.

FIGS. 13A-13B show biochemical characterization for all experimentalgroups. All values expressed as mean values. All ratios expressed asadipose-derived stem cells-to-chrondrocytes. FIG. 13A shows s-GAGcontent normalized to tissue wet weight (μg/mg). FIG. 13B shows s-GAGcontent normalized to DNA content (μg/mg). Error bars represent standarderror of the mean. s-GAG=sulfated glycosaminoglycan.DNA=dioxyribonuclease.

FIG. 14 shows a perspective top view of a multicomponent assembly earimplant tissue scaffold device prepared in accordance with certainaspects of the present disclosure having a plurality of hollow voidregions for receiving tissue samples therein.

Corresponding reference numerals indicate corresponding parts throughoutthe several views of the drawings.

DETAILED DESCRIPTION

Example embodiments are provided so that this disclosure will bethorough, and will fully convey the scope to those who are skilled inthe art. Numerous specific details are set forth such as examples ofspecific compositions, components, devices, and methods, to provide athorough understanding of embodiments of the present disclosure. It willbe apparent to those skilled in the art that specific details need notbe employed, that example embodiments may be embodied in many differentforms and that neither should be construed to limit the scope of thedisclosure. In some example embodiments, well-known processes,well-known device structures, and well-known technologies are notdescribed in detail.

The terminology used herein is for the purpose of describing particularexample embodiments only and is not intended to be limiting. As usedherein, the singular forms “a,” “an,” and “the” may be intended toinclude the plural forms as well, unless the context clearly indicatesotherwise. The terms “comprises,” “comprising,” “including,” and“having,” are inclusive and therefore specify the presence of statedfeatures, elements, compositions, steps, integers, operations, and/orcomponents, but do not preclude the presence or addition of one or moreother features, integers, steps, operations, elements, components,and/or groups thereof. Although the open-ended term “comprising,” is tobe understood as a non-restrictive term used to describe and claimvarious embodiments set forth herein, in certain aspects, the term mayalternatively be understood to instead be a more limiting andrestrictive term, such as “consisting of” or “consisting essentiallyof.” Thus, for any given embodiment reciting compositions, materials,components, elements, features, integers, operations, and/or processsteps, the present disclosure also specifically includes embodimentsconsisting of, or consisting essentially of, such recited compositions,materials, components, elements, features, integers, operations, and/orprocess steps. In the case of “consisting of,” the alternativeembodiment excludes any additional compositions, materials, components,elements, features, integers, operations, and/or process steps, while inthe case of “consisting essentially of,” any additional compositions,materials, components, elements, features, integers, operations, and/orprocess steps that materially affect the basic and novel characteristicsare excluded from such an embodiment, but any compositions, materials,components, elements, features, integers, operations, and/or processsteps that do not materially affect the basic and novel characteristicscan be included in the embodiment.

Any method steps, processes, and operations described herein are not tobe construed as necessarily requiring their performance in theparticular order discussed or illustrated, unless specificallyidentified as an order of performance. It is also to be understood thatadditional or alternative steps may be employed, unless otherwiseindicated.

When a component, element, or layer is referred to as being “on,”“engaged to,” “connected to,” or “coupled to” another element or layer,it may be directly on, engaged, connected or coupled to the othercomponent, element, or layer, or intervening elements or layers may bepresent. In contrast, when an element is referred to as being “directlyon,” “directly engaged to,” “directly connected to,” or “directlycoupled to” another element or layer, there may be no interveningelements or layers present. Other words used to describe therelationship between elements should be interpreted in a like fashion(e.g., “between” versus “directly between,” “adjacent” versus “directlyadjacent,” etc.). As used herein, the term “and/or” includes any and allcombinations of one or more of the associated listed items.

Although the terms first, second, third, etc. may be used herein todescribe various steps, elements, components, regions, layers and/orsections, these steps, elements, components, regions, layers and/orsections should not be limited by these terms, unless otherwiseindicated. These terms may be only used to distinguish one step,element, component, region, layer or section from another step, element,component, region, layer or section. Terms such as “first,” “second,”and other numerical terms when used herein do not imply a sequence ororder unless clearly indicated by the context. Thus, a first step,element, component, region, layer or section discussed below could betermed a second step, element, component, region, layer or sectionwithout departing from the teachings of the example embodiments.

Spatially or temporally relative terms, such as “before,” “after,”“inner,” “outer,” “beneath,” “below,” “lower,” “above,” “upper,” and thelike, may be used herein for ease of description to describe one elementor feature's relationship to another element(s) or feature(s) asillustrated in the figures. Spatially or temporally relative terms maybe intended to encompass different orientations of the device or systemin use or operation in addition to the orientation depicted in thefigures.

Throughout this disclosure, the numerical values represent approximatemeasures or limits to ranges to encompass minor deviations from thegiven values and embodiments having about the value mentioned as well asthose having exactly the value mentioned. Other than in the workingexamples provided at the end of the detailed description, all numericalvalues of parameters (e.g., of quantities or conditions) in thisspecification, including the appended claims, are to be understood asbeing modified in all instances by the term “about” whether or not“about” actually appears before the numerical value. “About” indicatesthat the stated numerical value allows some slight imprecision (withsome approach to exactness in the value; approximately or reasonablyclose to the value; nearly). If the imprecision provided by “about” isnot otherwise understood in the art with this ordinary meaning, then“about” as used herein indicates at least variations that may arise fromordinary methods of measuring and using such parameters. For example,“about” may comprise a variation of less than or equal to 5%, optionallyless than or equal to 4%, optionally less than or equal to 3%,optionally less than or equal to 2%, optionally less than or equal to1%, optionally less than or equal to 0.5%, and in certain aspects,optionally less than or equal to 0.1%.

In addition, disclosure of ranges includes disclosure of all values andfurther divided ranges within the entire range, including endpoints andsub-ranges given for the ranges.

As used herein, the terms “composition” and “material” are usedinterchangeably to refer broadly to a substance containing at least thepreferred chemical constituents, elements, or compounds, but which mayalso comprise additional elements, compounds, or substances, includingtrace amounts of impurities, unless otherwise indicated.

It should be noted that the figures set forth herein are intended toexemplify the general characteristics of methods, devices, andmaterials, among those of the present disclosure, for the purpose of thedescription of certain embodiments. These figures may not preciselyreflect the characteristics of any given embodiment, and are notnecessarily intended to fully define or limit specific embodimentswithin the scope of this disclosure.

Example embodiments will now be described more fully with reference tothe accompanying drawings.

In various aspects, the present disclosure provides ear implant devicesfor reconstructing tissue in a patient. The patient may be an animal,such as a mammal, including a human. The reconstructed tissue may beauricular tissue, including cartilage. In certain variations, the earimplant device is a multi-component implant assembly that comprises afirst component and a second distinct component that can be implantedvia surgery into or on the patient. In various aspects, the firstcomponent and the second component are tissue scaffolds that promotecell ingrowth. Thus, a first tissue scaffold component comprises aplurality of open pores configured to support cell growth and likewise,a second tissue scaffold component comprises a plurality of open poresconfigured to support cell growth. In various aspects, the first tissuescaffold component comprises a first biocompatible polymeric materialand the second tissue scaffold component comprises a secondbiocompatible polymeric material. Specific materials to be used in theimplant devices of the present technology that are biocompatible arepreferably biomedically acceptable. Such a “biomedically acceptable”material is one that is suitable for use with humans and/or animalswithout undue adverse side effects (such as toxicity, irritation, andallergic response) commensurate with a reasonable benefit/risk ratio.The first biocompatible polymeric material and the second biocompatiblepolymeric material may be the same or distinct compositions. In certainvariations, the first biocompatible polymeric material and/or the secondbiocompatible polymeric material may be composite materials having areinforcement phase or material distributed therein.

In certain embodiments, the ear implant devices of the presenttechnology comprise a biocompatible polymer, such as a biodegradablepolymer. The first biocompatible polymeric material and the secondbiocompatible polymeric material may independently comprise abiocompatible or biomedically acceptable polymer. The biocompatiblepolymer may be biodegradable or non-biodegradable. The term“biodegradable” as used herein means that the implant comprising thepolymer is slowly dissolved or disintegrated under physiologicalconditions in the human or other animal subject for a certain time andat some point only its degradation products are present in the body in adissolved or comminuted form. At this point, solid components orfragments of the implant either do not exist anymore or are so small asto be non-harmful or transported away by the subject's circulatorysystem. The degradation products are desirably substantially harmless inphysiological terms and lead to molecules that either occur naturally inthe human or other animal subject or can be excreted by the human orother animal subject.

Biodegradable polymers include polycaprolactone, polysebacic acid,poly(octaindiolcitrate), polydioxanone, polygluconate, poly(lactic acid)polyethylene oxide copolymer, modified cellulose, polyhydroxybutyrate,polyamino acids, polyphosphate ester, polyvalerolactone,poly-6-decalactone, polylactonic acid, polyglycolic acid, polylactides,polyglycolides, copolymers of the polylactides and polyglycolides,polye-caprolactone, polyhydroxybutyric acid, polyhydroxybutyrates,polyhydroxyvalerates, polyhydroxybutyrate-co-valerate,poly(1,4-dioxane-2,3one), poly(1,3-dioxane-2-one), poly-para-dioxanone,polyanhydrides, polymaleic acid anhydrides, polyhydroxy methacrylates,fibrin, polycyanoacrylate, polycaprolactone dimethylacrylates,poly-3-maleic acid, polycaprolactone butyl acrylates, multiblockpolymers from oligocaprolactonediols and oligodioxanonediols, polyetherester multiblock polymers from PEG and poly(butlylene terephthalates),polypivotolactones, polyglycolic acid trimethyl carbonates,polycaprolactone glycolides, poly(methyl glutamate), poly(DTH-iminocarbonate), poly(DTE-co-DT-carbonate), poly(bisphenolA-iminocarbonate), polyorthoesters, polyglycolic acid trimethylcarbonate, polytrimethyl carbonates, polyiminocarbonates,poly(N-vinyl)-pyrrolidone, polyvinyl alcohols, polyester amides,glycolized polyesters, polyphosphoesters, polyphosphazenes,poly[p-(carboxyphenoxy) propane], polyhydroxy pentanoic acid,polyanhydrides, polyethylene oxide propylene oxide, and combinationsthereof. In various embodiments, a preferred biodegradable biocompatiblepolymer that forms the ear implant device comprises, or consistsessentially of, polycaprolactone.

In various embodiments, the ear implant device comprising thebiodegradable biocompatible polymer allows the auricular tissue to growover and into the tissue scaffold and heal naturally. The implant maythen biodegrade or resorb in the subject or patient. Having the implantbiodegrade will not inhibit regrowth in adults or growth in children. Invarious embodiments, the ear implant device is designed to have adegradation time that coincides with the healing time that permitsregrowth of the defect in the patient. “Degradation time” refers to thetime for the ear implant device implanted to substantially and fullydissolve, disintegrate, or resorb. Depending upon the patient and thetime needed for recuperation and regeneration of the auricular tissue,the degradation time may be about 3 weeks to about 60 months (5 years),or about 2 months to about 40 months (3.33 years), or about 6 months toabout 36 months (3 years), or about 12 months to about 24 months (2years). As noted above, in certain embodiments, a preferredbiodegradable biocompatible polymer used to form the ear implant devicecomprises polycaprolactone, which desirably enables a degradation timeof 6 months to about 36 months (3 years) under normal physiologicalconditions when implanted in an animal subject/patient.

The term “non-biodegradable polymer” as used herein means that thebiocompatible or biomedically acceptable polymer forming the implantwill not dissolve in the human or animal subject. These polymers do notsubstantially resorb, dissolve or otherwise degrade after implantationin a human or animal subject, under typical physiological conditions.

In certain embodiments, the ear implant device of the present disclosureoptionally comprises a non-biodegradable biocompatible polymer. Suitablebiomedically acceptable non-biodegradable biocompatible polymers includepolyaryl ether ketone (PAEK) polymers (such as polyetherketoneketone(PEKK), polyetheretherketone (PEEK), andpolyetherketoneetherketoneketone (PEKEKK)), polyolefins (such asultra-high molecular weight polyethylene, which may be crosslinked, andfluorinated polyolefins such as polytetrafluorethylene (PTFE) or highdensity porous polyethylene), polyesters, polyimides, polyamides,polyacrylates (such as polymethylmethacrylate (PMMA)), polyketones,polyetherimide, polysulfone, polyurethanes, and polyphenolsulfones. Theear implant device may comprise multiple biocompatible polymers,including one or more biodegradable biocompatible polymers, one or morenon-biodegradable biocompatible polymers, and any combinations thereof.

The ear implant device of the present technology can further compriseone or more bioactive materials. More specifically, the firstbiocompatible polymeric material and the second biocompatible polymericmaterial may independently comprise a bioactive material. Depending onsuch factors as the bioactive material, the structure of the ear implantdevice, and the intended use of the implantable ear reconstructiondevice, the bioactive material may be coated on a surface of the firsttissue scaffold component or the second tissue scaffold component,coated or otherwise infused in the pores or openings of the first tissuescaffold component or the second tissue scaffold component, or mixed orcompounded within the first biocompatible polymeric material and thesecond biocompatible polymeric material of the ear implant device.

Bioactive materials can include any natural, recombinant or syntheticcompound or composition that provides a local or systemic therapeuticbenefit. In various embodiments, the bioactive material promotes healingand growth of an ear tissue resulting from a defect, including anotia,microtia, injuries or wounds resulting from trauma or surgery (such asoncologic surgical intervention). Bioactive materials among those usefulherein include cell adhesion factors, isolated tissue materials, growthfactors, peptides and other cytokines and hormones, pharmaceuticalactives, nanoparticles, and combinations thereof. Cell adhesion factorsinclude, for example, the RGD (Arg-Gly-Asp) sequence or the IKVAV(Ile-Lys-Val-Ala-Val) sequence. Growth factors and cytokines usefulherein include transforming growth factor-beta (TGF-β), including thefive different subtypes (TGF-β 1-5); bone morphogenetic factors (BMPs,such as BMP-2, BMP-2a, BMP-4, BMP-5, BMP-6, BMP-7 and BMP-8);platelet-derived growth factors (PDGFs); insulin-like growth factors(e.g., IGF I and II); fibroblast growth factors (FGFs), vascularendothelial growth factor (VEGF), epidermal growth factor (EGF) andcombinations thereof. Examples of pharmaceutical actives includeantimicrobials, antifungals, chemotherapeutic agents, andanti-inflammatories. Examples of antimicrobials include triclosan,sulfonamides, furans, macrolides, quinolones, tetracyclines, vancomycin,cephalosporins, rifampins, aminoglycosides (such as tobramycin andgentamicin), and mixtures thereof.

In certain variations, an ear implant device comprises a bioactivematerial in the form of a biomaterial that may be selected from thegroup consisting of: an isolated tissue material, a hydrogel,acellularized dermis, an acellularized tissue matrix, a composite ofacellularized dermis matrix and designed polymer, or a composite ofacellularized tissue matrix and designed polymer, and combinationsthereof. An isolated tissue material may include an autologous orallogeneic tissue sample (such as an explant harvested in the patient bya punch biopsy to provide tissue sample). In other aspects, an isolatedtissue material may include isolated or cultured cells (such aschondrocyte cells, hemopoietic stem cells, mesenchymal stem cells, suchas adipose-derived mesenchymal stem cells, endothelial progenitor cells,fibroblasts, reticulacytes, and endothelial cells), whole blood andblood fractions (such as red blood cells, white blood cells,platelet-rich plasma, and platelet-poor plasma), collagen, fibrin,acellularized dermis, and the like. In one embodiment, the isolatedtissue biomaterial may comprise a combination of porcine adipose-derivedstem cells and/or bone marrow derived or induced pluoripotent stem cellswith chondrocytes, which may be combined at ratios of about 1:1 to 10:1.Hydrogels are materials formed from lightly-crosslinked networks ofnatural or synthetic polymers, such as saccharides, which have highwater contents such as 90% weight per volume or greater. Hydrogelcrosslinking can be achieved by various methods including ionic,covalent chemical, or UV-initiated chemical crosslinking. Hydrogels usedin the present disclosure are preferably biocompatible. Hydrogels may beformed from hyaluronic acid/hyaluronan, sodium alginate, polyethyleneglycol (PEG), polyethylene glycol diacrylate (PEGDA), 2-hydroxyethylmethacrylate (HEMA)/poly(2-hydroxyethyl methacrylate) (pHEMA),polymethyl methacrylate (PMMA), polyacrylic acid, chitosan, poly(aminoacids), poly(N-isopropylacrylamide) (PNIPAM), collagen, gelatin,fibronectin, chondroitin sulfate, surfactant gels (having greater thanabout 20% weight per volume poloxamers (e.g., commercially available asPLURONIC™ and BRIJ™), polydimethylsiloxane (PDMS) or dimethicone, epoxy,polyurethane, and the like. In one embodiment, a suitable hydrogel basedbiomaterial may comprise hyaluronic acid and Type I collagen. In certainaspects, an implantable ear device may have a biomaterial disposed onone or more surfaces that will contact tissue in the patient uponimplantation of the ear implant device.

In certain variations, the first tissue scaffold component consistsessentially of the first biocompatible polymeric material and anoptional bioactive agent/biomaterial and the second tissue scaffoldcomponent consists essentially of the biocompatible polymeric materialand an optional bioactive agent/biomaterial prior to implantation in thepatient.

The present disclosure thus contemplates scaffold based tissueengineering for ear reconstruction. The ear implant assembly may beimplanted in a patient selected from prefabricated implant deviceshaving common sizes and dimensions or may be customized to a patient byusing an image-based design approach to tailor the design to a specifichuman or animal subject. Where the ear implant device is manufacturedfor a specific patient, it provides a personalized, customizablesolution for several possible applications in ear reconstruction,including reconstruction of microtia, anotia, or congenital deformity,reconstruction in oncologic resection, and reconstruction in trauma orblast injury, by way of example. Such techniques provide the ability toincorporate age, gender, and ethnic specific properties to these earimplant devices. The image-based design approach uses medical images orother data that is specific to the patient to customize the size of theear implant device. Scaffold implants can be produced from extrapolationof ear defects or virtual repair of congenital malformations. Also,mirrored scaffolds allowing perfect symmetry in repair can be produced.

With reference to FIGS. 1A-1B, first specific medical images and/orparameters are obtained from one or more imaging systems such ascomputed tomography (CT), a CT-fluoroscopy, fluoroscopy, magneticresonance imaging (MRI), ultrasound, positron emission tomography (PET)and X-Ray systems or any other suitable imaging systems. In certainaspects, an ear implant scaffold may be produced from a laser or light3D scan, multipod photography, or pre-existing CT/MRI scan of thepatient.

The medical image data and/or parameters received from the imagingsystem provide a two-dimensional (2D), three-dimensional (3D) orfour-dimensional (4D) model of an anatomical structure, system or regionof the patient, here the ear region of the patient. The image-baseddesign of the 2D, 3D or 4D model may be created using MATLAB®,Mathematica®, or other computer-aided-designed (CAD) software designprograms known in the art. For converting the design into a usableformat for rapid prototyping and computer-aided manufacturing, a STLfile format may be created. This file format is supported by manysoftware packages such as Mimics® by Materialise, MATLAB®, IDL, andAmira®. More specifically, Digital Imaging and Communications inMedicine (DICOM) data is shown for the anatomic structure of interest(e.g., an ear region) of a patient in FIG. 1A and used to generate athree-dimensional model of the structure shown in FIG. 1B.

This 2D, 3D or 4D model of the ear implant device of the presenttechnology may then be used to manufacture the ear implant device. Theimplant device may be made by a variety of suitable methods, includingmethods comprising solid free-form fabrication (SFF) techniques, such aslaser sintering, stereolithography, 3D printing, injection molding andthe like. In various embodiments, the preferred method is an additivemanufacturing process of laser sintering. Laser sintering is a processinvolving the construction of a three-dimensional article by selectivelyprojecting a laser beam having the desired energy onto a layer ofparticles of the polymer material to be sintered. The laser sinteringprocess can be paired with medical image data and/or parameters receivedfrom the imaging system for producing a customized ear implant device ofthe present technology. The model in FIG. 1B is thus converted into aporous structure using negative Boolean operations shown in FIG. 1C andmanufactured from polycaprolactone using an additive manufacturingprocess, such as selective laser sintering three-dimensional (3D)printer.

In FIG. 1D, the pores of the bioresorbable scaffold are then seeded withcells suspended in a hyaluronic acid/collagen hydrogel (shown in FIG.1E) prior to implantation. FIG. 1F shows an ear implant afterimplantation and tissue growth and reconstruction. In this manner, thepresent disclosure contemplates forming mirrored tissue scaffoldsallowing perfect symmetry in repair. Such techniques can produce patientspecific anatomic soft tissue implants and tissue engineering scaffoldsthat can reproduce complex craniofacial structures with high fidelity.

FIGS. 2A-2B show a computer-aided design (CAD) and a three-dimensionalprinting process for production of a porous bioresorbable tissueengineering scaffold. In FIG. 2A, a rendering of stereolithography(.STL) file for an example cylindrical tissue engineering scaffold 50having a plurality of open and interconnected pores 52 with a 2.7 mmspherical pore internal microarchitecture prepared in accordance withcertain aspects of the present disclosure is shown. This STL file canform a final tissue engineering scaffold manufacturing via selectivelaser sintering three-dimensional printing technique of a biocompatiblepolymer, such as polycaprolactone. Scaffold features as small as about70 μm can be successfully reproduced with this approach. FIG. 2B showsfinal tissue engineering scaffolds after cell seeding in a hyaluronicacid/collagen hydrogel implanted into four randomized quadrants in asubcutaneous pocket on the dorsum of an athymic rat.

Referring to FIG. 3 , an ear implant device 20 is shown that is amulticomponent assembly that serves as a tissue scaffold when implanted.The ear implant device 20 includes a first tissue scaffold component 22that defines a central void region 24 and at least a portion of an outerear framework 26 of the patient. By outer framework, it is meant that atleast a portion of the auricle of the ear, including helix, lobule,antitragus, and/or tragus regions, may be reconstructed via tissuegrowth. In certain variations, the outer ear framework may be anenclosed ring structure defining the central void region 24. In othervariations, the outer ear framework may have a C-shape with a narrowopening that defines the central void region 24. The first tissuescaffold component 22 comprises a first biocompatible polymeric materialhaving a first plurality of open pores 28 configured to support cellgrowth. A first plurality of open pores 28 that can support cell growthmay have an average pore diameter of greater than or equal to about 70μm to less than or equal to about 8 mm. The plurality of open poresdesirably is interconnected and provides fluid flow between the pores topermit flow of nutrients and fluids therebetween. The plurality of openpores may be regularly distributed in a repeating or random pattern, butin certain preferred aspects, are regularly and evenly distributedwithin the scaffold. The pores may have a variety of shapes, but incertain variations, may be a round shape, such as spherical. Thescaffold may optionally have a porosity of greater than or equal toabout 50% by volume to less than or equal to about 95% by volume voidsor open pores, optionally greater than or equal to about 50% by volumeto less than or equal to about 70% by volume open pores. Such poreproperties can facilitate and support good cell and tissue ingrowth whenthe scaffold device is implanted into the patient.

The ear implant device 20 also includes a second tissue scaffoldcomponent 30 defines a base portion. After implantation into thepatient, the second tissue scaffold component 30 seats within a portionof the central void region 24 of the first tissue scaffold component 22,so that the second tissue scaffold component 30 is secured to the firsttissue scaffold component 22 and thus defines an ear implant scaffoldassembly. The base portion may define at least a part of the triangularfossa, antihelix, and/or concha regions of the ear may be recreated andin certain variations and the base portion will recreate the centraltissue structures inside the ear outer framework, although in certainembodiments, the second tissue scaffold component may also extend into aportion of the outer ear framework, for example, extending into thehelix region of the ear. The second tissue scaffold component 30comprises a second biocompatible polymeric material having a secondplurality of open pores 32 configured to support cell growth. The secondplurality of open pores 32 can have the same properties as the firstplurality of open pores 28 discussed above. As can be seen, the earimplant has a width of approximately 30 mm and a height of approximately72 mm, although these dimensions are merely exemplary and non-limiting.

Non-limiting examples of suitable dimensions for ear implants formed inaccordance with certain aspects of the present disclosure are asfollows. The ear implant device, for example, the assembled componentsin a multicomponent implant, may have a width ranging from greater thanor equal to about 20 mm to about 50 mm and a height ranging from greaterthan or equal to about 35 mm to about 75 mm.

For purposes of comparison, a high density porous polyethylene MEDPOR™ear implant 40 that is commercially available from Stryker Corp. isshown for comparison in FIG. 1 . Prior to implantation, the componentsare fused to one another. As noted previously, MEDPOR™ ear implants arenot tissue scaffolds and do not have the capacity for growth, because nocell seeding or tissue ingrowth occurs such high density porouspolymeric implants. Further, only a single sized ear construct isavailable to meet the needs of the wide range of pediatric and adultpatients needing reconstruction. The rigidity of the MEDPOR™ ear implantcombined with other shortcomings in the design result in high levels ofcomplications, fracture, exposure, extrusion, and infection of porouspolyethylene device after implantation into patients.

Referring to FIGS. 4A-4D, a multicomponent ear implant assembly 60 isshown that serves as a tissue scaffold when implanted in a patient (bestseen in FIGS. 4C-4D). Each figure shows a computer-assisted-model of thecomponent and the laser sintered scaffold formed from the model. Asshown in FIG. 4A, the ear implant assembly 60 includes a first tissuescaffold component 62 that defines a central void region 64 and at leasta portion of an outer ear framework 66 of the patient. The first tissuescaffold component 62 comprises a first biocompatible polymeric materialhaving a first plurality of open pores 68 configured to support cellgrowth. The first plurality of open pores 68 have a tetrahedral unitshape and may have the dimensions and properties described previouslyabove in the context of FIG. 3 . The first tissue scaffold component 62further comprises a first interlock member 70. As shown, the firstinterlock member 70 defines a rectangular shaped void 72.

The multicomponent ear implant assembly 60 also includes a second tissuescaffold component 80 that defines a base portion of the assembly 60 asshown in FIG. 4B. After implantation into the patient, the second tissuescaffold component 80 seats within a portion of the central void region64 of the first tissue scaffold component 62, so that the second tissuescaffold component 80 is secured to the first tissue scaffold component62. The second tissue scaffold component 80 comprises a secondbiocompatible polymeric material having a second plurality of open pores82 configured to support cell growth. The second plurality of open pores82 can have the same properties as the first plurality of open pores 68discussed above or in the context of FIG. 3 . The second tissue scaffoldcomponent 80 further comprises a second interlock member 84. The secondinterlock member 84 may be in a shape of a rectangular projection.Notably, other shapes complementary with the opposing interlock memberare also contemplated. The first interlock member 70 on the first tissuescaffold component 62 and the second interlock member 84 on the secondtissue scaffold component 80 are coupled together and secure the firsttissue scaffold component 62 to the second tissue scaffold component 80,as shown in FIGS. 4C and 4D. The first and second interlock features maytogether define a dove tail interlock assembly, an offset snap assembly,have contrapositive interlocking shapes, and the like to secure thecomponents together in the assembly.

In certain aspects, a multicomponent ear implant assembly 60 like thatshown in FIGS. 4A-4D may be used in a multistage implantation procedure.In certain variations, the first tissue scaffold component 62 may firstbe implanted in the patient to facilitate ingrowth of cells and tissueover and within the scaffold to define an outer ear framework. The outerear framework may have a relatively low profile without significantelevation. After a sufficient amount of tissue growth has occurred, thena second surgical procedure may implant the second tissue scaffoldcomponent 80 into the patient. The first interlock member 70 and thesecond interlock member 84 are coupled together during this procedure tosecure the first tissue scaffold component 62 to the second tissuescaffold component 80. Notably, the design of the ear implant shown inFIGS. 4A-4D and more specifically, the second interlock member 84 of thesecond tissue scaffold component 80 is configured to elevate the entireimplant assembly 60 and serves as a “kickstand” or “prop” of theassembly.

In this manner, the introduction of the second tissue scaffold component80 serves to raise a height and profile of the implant assembly 60, thusfacilitating profiling to approximate natural ear shape in the secondstage of growth within the tissue scaffold. The low profile of the firsttissue scaffold component 62 reduces the incidence of dehiscence. Then,the second tissue scaffold component 80 is implanted underneath theinitial conformational shape of the auricle corresponding to the firsttissue scaffold component 62 and thus elevates the auricle to obtainfull projection. The secondary tissue scaffold component 80 can snapinto the primary implanted first tissue scaffold component 62. Such amultistage implantation procedure may be particularly advantageous wherethe ear size may change (for example, in a growing child) or where thetissue at the target site is too thin or delicate to accommodate theheight of the entire assembly in a single implantation procedure. Inalternative aspects, the components of the multicomponent ear implantassembly 60 may be secured prior to an initial implantation surgery andimplanted as an entire assembly in one procedure.

In certain variations, second interlock member 84 rectangular projectionmay comprise a tissue expansion device that may facilitate additionaltissue expansion after implantation. It should be noted that otherregions of the implant assembly 60 may have a tissue expansion deviceand it is not limited to the rectangular projection of the secondinterlock member 84. For example, the tissue expander may be a bladderor balloon into which gas or fluid may be serially injected to graduallyexpand the expansion device and surrounding tissue. Similarly, thetissue expansion device may be an osmotic expansion device thatgradually increases in volume over time to facilitate tissue expansion.As noted above, where the second interlock member 84 is a kickstand thatserves as a way to increase height and profile of the implant assembly60, such tissue expansion may increase the height of the overall implantassembly 60. In various aspects, the present disclosure provides amulticomponent ear implant assembly that provides an ability to projectwith or without a tissue expansion device. When used, the accompanyingtissue expansion mechanism allows for calculated projection with gradualexpansion and growth.

In variations where a multistage implantation process is contemplated,the first tissue scaffold component 62 may further comprise a removableguard 88 that serves to protect one or more edges of the implanted firsttissue scaffold component 62. Thus, the removable guard 88 may cover andprotect the edges of the rectangular projection of the second interlockmember 84 during initial implantation in the patient. The removableguard 88 may be formed of a biocompatible material, such as silicone.However, the removable guard 88 is configured to be removed during asubsequent second procedure, when the second tissue scaffold component80 is implanted into the patient. The removable guard may have a knob orother feature that facilitates removal by a surgeon. The removable guard88 protects the edge from tissue growth and provides a clean andunobstructed edge so that the first tissue scaffold component 62 and thesecond tissue scaffold component 80 can couple with one another withoutobstruction or interference. Thus the removable guard 88 may beconsidered to be a silicone place holder in first stage tissue scaffoldthat provides a fresh edge upon removal and facilitates precise fit ofsubsequent stage scaffold module. It should be noted that any edge orsurface of an implantable device in accordance with the presentdisclosure that may require protection during implantation can have aremovable guard disposed thereon. Further, other shapes and sizes arecontemplated from those shown in FIG. 4D.

Referring to FIGS. 5A-5C, another multicomponent ear implant assembly100 is shown that serves as a tissue scaffold when implanted in apatient (best seen in FIG. 5B). In FIG. 5A, the ear implant assembly 100includes a first tissue scaffold component 102 that defines a centralvoid region 104 and at least a portion of an outer ear framework 106 ofthe patient. The first tissue scaffold component 102 comprises a firstbiocompatible polymeric material having a first plurality of open pores(not shown in the models of FIGS. 5A-5C) configured to support cellgrowth. The first plurality of open pores may have the dimensions andproperties described previously above. The first tissue scaffoldcomponent 102 further comprises at least one expandable opening 108. Theexpandable opening 108 may serve as a first interlock member. As shownin FIG. 5C, the expandable opening 108 expands the body of the scaffoldcorresponding to the body of the outer ear framework 106 outwards in thedirection of the arrows.

The multicomponent ear implant assembly 100 also includes a secondtissue scaffold component 120 that defines a base portion of theassembly 100 as shown in FIG. 5B. After implantation into the patient,the second tissue scaffold component 120 seats within a portion of thecentral void region 104 of the first tissue scaffold component 102, sothat the second tissue scaffold component 120 is secured to the firsttissue scaffold component 102. The second tissue scaffold component 120comprises a second biocompatible polymeric material having a secondplurality of open pores configured to support cell growth. The secondplurality of open pores can have the same properties as discussed above.The second tissue scaffold component 120 further comprises a secondinterlock member 124. The second interlock member 124 may be in a shapeof a rectangular projection that is dimensioned to fit within theexpandable opening 108 of the first tissue scaffold component 102.Notably, other shapes complementary with the opposing interlock memberare also contemplated. The expandable opening 108 on the first tissuescaffold component 102 and the second interlock member 124 on the secondtissue scaffold component 120 are coupled together, as shown in FIG. 5B.Other interlock features may also be used, including those describedpreviously above. Notably, the presence of the second tissue scaffoldcomponent 120 seated within the void region 104 of the first tissuescaffold component 102 forces the outer ear framework 106 outwards.

In certain aspects, a multicomponent ear implant assembly 100 like thatshown in FIGS. 5A-5C may be used in a multistage implantation procedure.In certain variations, the first tissue scaffold component 102 may firstbe implanted in the patient to facilitate ingrowth of cells and tissueover and within the scaffold to define an outer ear framework. After asufficient amount of tissue growth has occurred, then a second surgicalprocedure may implant the second tissue scaffold component 120 into thepatient. The second tissue scaffold component 120 is seated within thevoid region 104 and the second interlock member 124 is disposed withinthe expandable opening 108 of the outer ear framework 106, serving tocouple the first tissue scaffold component 102 to the second tissuescaffold component 120 during the procedure. Notably, the design of theear implant shown in FIGS. 5A-5C uses the second tissue scaffoldcomponent 120 to expand the outer ear framework 106 of the first tissuescaffold component 102 as a “table leaf insert” and can also elevate theentire implant assembly 100. In this manner, the introduction of thesecond tissue scaffold component 120 serves to expand the first tissuescaffold component 102 and optionally raise a height and profile of theimplant assembly 100, thus facilitating profiling to approximate naturalear shape in the second stage of growth within the tissue scaffold. Sucha multistage implantation procedure may be particularly advantageouswhere the ear size may change (for example, in a growing child) or wherethe tissue at the target site is too thin or delicate to accommodate theheight of the entire assembly in a single implantation procedure. Incertain aspects, the second tissue scaffold component 120 can besubsequently replaced with a longer table leaf to further facilitateexpansion and growth. In alternative aspects, the components of themulticomponent ear implant assembly 100 may be secured prior to aninitial implantation surgery and implanted as an entire assembly in oneprocedure.

Thus, a two staged 3D printed ear scaffold implantation process canallow for improved outcomes and decreased complications when compared toa single staged implant for animal implantation. Without being limitedto any particular theory, the hypothesis is that a two staged scaffolddesign allows for more robust tissue ingrowth and vascularization of thesuperficial ear scaffold, which upon elevation in the second stage willmitigate extrusion, infection, and scaffold exposure observed in singlestage scaffold implants. Ear scaffold implants having a mechanism ofmodular scaffold expansion are thus contemplated. Such scaffolds providethe ability to predictably enlarge the ear scaffold at a second stagesurgery, while simultaneously providing ear projection. The ability toprovide for expansion of an ear framework is not presently possible, andis a unique design capability imparted by 3D printing of the scaffoldimplant assemblies prepared in accordance with certain aspects of thepresent disclosure.

In this embodiment, the mechanism of expansion emulates expansionprovided by a table center leaf, with precision of increase in dimensionand interlocking mechanism provided by the precision of the 3D printingdesign and process. The result of this design provides a novel featureto ear reconstruction—earlier implantation and the appearance of eargrowth commensurate with growth of the child. Moreover, due to theunique nature of patient specific, 3D printed scaffolds allow forunparalleled control and match to the contralateral ear at both theinitial framework implantation, and second stage elevation. 3D printedmodular scaffolds can consistently increase ear scaffold dimension inthe second stage surgery, giving the appearance of growth after thefirst stage surgery by at least about 20%.

FIGS. 6 and 7 show a first tissue scaffold component 130 that defines acentral region 132 and an outer ear framework 134 of a patient. To theextent that the features of the first tissue scaffold component 130 arethe same as the previous embodiments described, for brevity, they willnot be repeated herein. The first tissue scaffold component 130comprises a plurality of open pores 136 configured to support cellgrowth and having a tetrahedral unit shape with dimensions andproperties previously described. An internal open channel 140 isdisposed within the first tissue scaffold component 130 and is in fluidcommunication with the plurality of pores 136. As best seen in FIG. 7 ,the first tissue scaffold component 130 includes a drain port 142 thatis in fluid communication with the internal open channel 140. In thismanner, in certain variations, an external suction device, such as aconduit having negative pressure, can be attached to the drain port 142to facilitate removal of fluids from the internal open channel 140 andpores 136 as needed. Suction pathways allow the scaffold implant toserve as its own suction port, thus providing optimal soft tissueadherence and minimization of hematoma or seroma formation.

In certain other variations like that in FIG. 14 , a multicomponent earimplant assembly 150 includes both a first tissue scaffold component 152that defines a central void region 154 and at least a portion of anouter ear framework 156 of a patient and a second tissue scaffoldcomponent 160 that defines a body portion and seats within the centralvoid region 154 of the first tissue scaffold 152. Both the first tissuescaffold component 152 and the second tissue scaffold component 160 maycomprise biocompatible polymeric materials and have a plurality of openpores configured to support cell growth. The first tissue scaffoldcomponent 152, the second tissue scaffold component 160, or both mayinclude one or more hollow features 170 through a body of the scaffold.The hollow feature(s) 170 are configured to receive a tissue sample. Incertain variations, the hollow feature(s) 170 is cylindrical with around or circular cross-section, although other shapes includingsemilunar, elliptical, and the like are contemplated. The hollow featuremay have a width or diameter of greater than or equal to about 1 mm toless than or equal to about 12 mm. In various aspects, the hollowfeature is significantly larger than a plurality of pores in thescaffold, for example, an average width or diameter of the hollowfeature may be at least 100% greater than an average dimeter pore size.The hollow feature may extend from one surface into the scaffold for apredetermined distance or may fully extend from one surface through anopposite surface to form an aperture through the scaffold. In certainembodiments, the hollow feature may be designed with back stop rim orledge that prevents displacement of the tissue sample during handlingand implantation into the patient. As shown in FIG. 14 , the hollowfeatures 170 include a first plurality of hollow features 172 in thefirst tissue scaffold component 152 having a first diameter. The hollowfeatures 170 also include a second plurality of hollow features 174 inthe second tissue scaffold component 160 that have a second diameter.The first diameter and second diameter are distinct from one another. Asshown, the second diameter is greater than the first diameter.

One or more tissue samples may be harvested from the patient (or fromanother source of tissue) via a punch biopsy tool or other technique.The punch biopsy tool may be dimensioned to provide tissue samples thatwill seat within the hollow feature. In certain variations, the tissueharvested is cartilage tissue. After harvesting, the tissue sample maybe disposed within the hollow features of the scaffold implant. Thus,the scaffold implant may be seeded by implanting cartilage punches priorto implantation into the patient. Custom punch biopsy designs andaccompanying scaffold inserts allow for rapid and precise harvest anddistribution of tissue within the scaffold when the tissue sample isdisposed in the one or more hollow features. The scaffold design allowsfor precise placement and distribution of cartilage punches. In certainvariations, a plurality of hollow features may be provided in variousregions of the first tissue scaffold component and/or the second tissuescaffold component. The size and distribution of hollow features foraccepting cartilage punch biopsy inserts can be determined based onFinite Element Analysis to guide relief of overlying soft tissue strainand vascular compromise. In certain variations, the hollow features thataccept prefabricated inserts are offset, so as to allow the tissuesample to protrude from the tissue scaffold, ranging from greater thanor equal to about 50 μm to less than or equal to about 10 mm, based onFinite Element Analysis guiding relief of overlying soft tissue strainand vascular compromise. Porous scaffold designs radiating fromprefabricated hollow features that accept punch biopsy insertsfacilitate cellular, paracrine, and autogenous growth factordissemination. Further, an eluting periinsert component allows forgradual dissolution of the tissue sample/cartilage punch, furtherfacilitating cellular outgrowth, paracrine influence. Thus, a macroporehollow feature configured to accept cartilage punches advantageouslypromotes cellularization of the tissue scaffold, while minimizingoperative time and accelerating translation.

In yet other variations, a multicomponent ear implant assembly includesboth a first tissue scaffold component that defines a central voidregion and at least a portion of an outer ear framework of a patient anda second tissue scaffold component that defines a body portion and seatswithin the central void region of the first tissue scaffold. Both thefirst tissue scaffold component and the second tissue scaffold componentmay comprise biocompatible polymeric materials and have a plurality ofopen pores configured to support cell growth. The first tissue scaffoldcomponent, the second tissue scaffold component, or both may include atleast one Luer connector. The Luer connector is compatible with acomplementary Luer connector, for example, on a syringe to form a Luerlock providing a fluid seal. The Luer connector may be in fluidcommunication with the plurality of pores. Optionally, one or moreinternal channels may also be disposed within the first tissue scaffoldcomponent and/or the second tissue scaffold component and in fluidcommunication with the Luer connector. A Luer connector can form a Luerlock assembly with an external syringe or injector, allowing forexternal materials to be injected within the pores or hollow featureswithin the implant scaffold. For example, hydrogels, solutions,nanoparticles, growth factors, cells, tissue infusions, and the like maybe injected into the ear implant device via the Luer lock.

In certain variations, an implant assembly for reconstruction ofauricular tissue in a patient is provided, where the implant comprises afirst tissue scaffold component and a second tissue scaffold component.The first tissue scaffold component comprises a first biocompatiblepolymeric material having a first plurality of open pores configured tosupport cell growth. The first tissue scaffold component defines acentral void region and at least a portion of an outer ear framework ofthe patient after implantation. The second tissue scaffold componentcomprises a second biocompatible polymeric material having a secondplurality of open pores configured to support cell growth. The secondtissue scaffold component defines a base portion and after implantationinto the patient. The second tissue scaffold component seats within thecentral void region of the first tissue scaffold component, so that thesecond tissue scaffold component is secured to the first tissue scaffoldcomponent.

In certain embodiments, the first plurality of open pores in the firsttissue scaffold component has a first pore density in a first region.The pore density thus translates to a first rigidity level. The firsttissue scaffold component also has a second region having a second poredensity distinct from the first pore density. The second pore densitythus relates to the second region having a second rigidity leveldistinct from the first rigidity level. Alternatively or in addition,the second plurality of open pores in the second tissue scaffoldcomponent may have a first pore density in a first region having a firstrigidity level, while the second tissue scaffold component has a secondpore density distinct from the first pore density in a second region.The second region thus has a second rigidity level distinct from thefirst rigidity level. In this manner, the implant devices have atailored scaffold porosity, which provides the capability of forminghybrid or gradated scaffold pores within the ear scaffold implant. Thepore density and/or pore architecture imparts strength and rigidity indesired regions of the implant, for example, at foundational subunits(e.g., concha cymba, and concha cavum regions of the ear). Similarly,the pore density and/or porous architecture can impart flexibility indesired regions of the implant, for example, at foundational subunitsadjacent to soft tissue interfaces (e.g., helix, antihelix, andintertragal complex regions of the ear). Thus, the implant scaffoldstiffness can be mediated by microstructure design, providing theability to finely balance a construct design that is robust enough toprevent contracture, yet with a modulus low enough to reduce the risk ofmechanical strain on vasculature with subsequent construct extrusionthrough overlying soft tissue.

In certain aspects, differences in porosity within the tissue implantcan desirably impart gradated permeability to facilitate fluid flowwithin the pores of the implant in a predetermined direction. Moreover,controlling the porous characteristics of the tissue scaffolds caninfluence differentiation of pluoripotent stem cells, which is believedto provide an optimal environment for cartilage or bone. Thus, the poresmay have different sizes and pore density or different shapes within thescaffold implant design from one region to another. High internalpermeability and low peripheral permeability allow for low impedence andhigh homogeneity with external materials introduced into the implant,such as hydrogels, solutions, nanoparticles, growths factors, cells,tissue infusions, and any combinations thereof.

In other variations, the first tissue scaffold component may have anaverage porosity level that is distinct from an average porosity levelin the second tissue scaffold, which may provide different levels ofrigidity and/or flexibility in the first tissue scaffold component ascompared to the second tissue scaffold component. The first plurality ofopen pores in the first tissue scaffold component may thus have a firstaverage pore density corresponding to a first average rigidity level.The second plurality of open pores in the second tissue scaffoldcomponent has a second average pore density corresponding to a secondaverage rigidity level distinct from the first average rigidity level.In certain variations, the second average rigidity level in the secondtissue scaffold is greater than the first average rigidity level in thefirst tissue scaffold, so that the first tissue scaffold component hashigher flexibility. The second tissue scaffold component having a higherrigidity in the central region of the implant provides structuralsupport to the auricular tissue as it regrows and helps to facilitateprojection and expansion, while minimizing or preventing collapse of thereconstructed tissue.

In certain aspects, the auricular reconstruction scaffold implantsprepared in accordance with the present disclosure provides one or moreof the following benefits: (1) ease and precision to reconstruct complexear geometry, (2) personalization to replicate an individual's auricularanatomic structure with the ability of expansion giving the appearanceof growth, (3) resistance to contraction, for example, allowingmeticulous control of mechanical properties, specifically, to impartsufficient stiffness allowing resistance against contracture, whilebalancing compliance to prevent dehiscence, (4) preventing exposure dueto dehiscence with subsequent infection, (5) potential to serve as aplatform to deliver biologics for tissue growth, (6) customization toimplement different staged procedures and special surgical requirementssuch as surgical drains, and (7) facilitating tissue engineering in alow cost, low resource environment.

In certain variations, the present disclosure contemplates a kit thatmay have a plurality of distinct implant assemblies formed in accordancewith the present teachings as described in the various embodimentsabove. Each ear implant device may be prefabricated with a range ofcertain predetermined properties, such as size, rigidity/flexibility,and the like or with different features, such as internal channels,drain ports for suction and draining, hollow regions for receivingtissue samples, Luer connectors, implants formed from resorbablebiocompatible materials or non-resorbable biocompatible materials,bioactive agents or biomaterials, and the like. For example, suchimplant assemblies in the kit may have different dimensions or sizes toprovide a variety of options of available ear implants, so that themedical practitioner can select an appropriately sized ear implant forthe specific patient. In one aspect, the present disclosure contemplatesa kit comprising at least one implant assembly having a first size andat least one implant assembly having a second size distinct from thefirst size. The kit may include other components, such as tools like atissue harvesting tool (e.g., one or more punch biopsy tools), a syringethat may interface with a Luer connector on the implant to form Luerlock, and the like.

In certain other variations, a method for reconstructing auriculartissue in a patient is provided. The method may include implanting afirst tissue scaffold component in an ear region of the patient. Thefirst tissue scaffold component comprises a first biocompatiblepolymeric material having a first plurality of open pores that supportcell growth after the implanting. The first tissue scaffold componentdefines a central void region configured to receive a second tissuescaffold component and at least a portion of an outer ear framework ofthe patient. The second tissue scaffold component seats within thecentral void region of the first tissue scaffold component and defines abase portion of an implant assembly comprising the first tissue scaffoldcomponent and the second tissue scaffold component.

In certain variations, the implanting may be a first implanting andafter auricular tissue has grown over the first tissue scaffoldcomponent in the patient, the method further comprises a secondimplanting of the second tissue scaffold component in the central voidregion to secure the second tissue scaffold component to the firsttissue scaffold component. The second tissue scaffold componentcomprises a second biocompatible polymeric material having a secondplurality of open pores that support cell growth and increases anelevation of the implant assembly.

In other variations, the first tissue scaffold component furthercomprises a removable guard to protect at least one edge of the firsttissue scaffold component and prior to the second implanting of thesecond tissue scaffold, the removable guard is removed.

In yet other variations, the first tissue scaffold component furthercomprises a first interlock member and the second tissue scaffoldcomponent further comprises a second interlock member. The firstinterlock member and the second interlock member are coupled together tosecure the first tissue scaffold component to the second tissue scaffoldcomponent and the first interlock member and the second interlock membertogether define a dove tail interlock assembly or an offset snapassembly.

In certain variations, at least one of the first tissue scaffoldcomponent or the second tissue scaffold component further comprises atleast one hollow feature configured to receive a tissue sample. Themethod includes harvesting the tissue sample from the patient andinserting it into the at least one hollow feature prior to the firstimplanting, the second implanting, or both the first implanting and thesecond implanting.

In certain other variations, the first tissue scaffold componentcomprises at least one expandable opening and the second tissue scaffoldcomponent seats with the central void region to facilitate outwardexpansion of the first tissue scaffold component after the secondimplanting of the second tissue scaffold component in the patient.

In yet other variations, the second tissue scaffold component comprisesa rectangular projection and the first tissue scaffold component definesa rectangular void to receive the rectangular projection, wherein therectangular projection is configured to elevate the implant assembly.

In further variations, the rectangular projection comprises a tissueexpansion device and the method further comprises expanding tissue byadjusting the tissue expansion device.

In certain variations, the first tissue scaffold component furthercomprises a drain port and an internal channel in fluid communicationtherewith. The method further comprises connecting the drain port with aconduit under negative pressure to remove fluids within the internalchannel.

Various embodiments of the inventive technology can be furtherunderstood by the specific examples contained herein. Specific Examplesare provided for illustrative purposes of how to make and use thecompositions, devices, and methods according to the present teachingsand, unless explicitly stated otherwise, are not intended to be arepresentation that given embodiments of this invention have, or havenot, been made or tested.

EXAMPLES Example A

Scaffolds are created using image-based hierarchical design and lasersintering methods. The manufacturing methods begin with the possibilityof using photographically or radiographically obtained patient specificimaging data to 3D print unique custom scaffolds, an improvement overprefabricated porous polyethylene implants. Furthermore, laser sinteringallows for the ability to impart meticulous architecture of the poreswithin the already intricately designed scaffold.

Hierarchical image designs are created separately for the global earstructure. Designs are represented by a density distribution within avoxel format, similar to the way 3D images are represented by densitydistributions within a voxel dataset. Separate voxel design datasets arecreated for the anatomic structure, based on an actual patientradiologic data. Different pore structures are created by generatingeither periodic or random geometries—such as spheres or cylinders usingdensity distributions in voxel data structures created by speciallywritten MATLAB™ codes. Both the anatomic and porous designed voxelstructures are then converted into a triangular surface .STLrepresentation. A final scaffold design is created by mapping a porousarchitecture STL file into the appropriate location of the anatomicdataset (also represented as a .STL file after conversion in thecommercial software MIMICS™ by Materialise). A porous architecture,either periodic spherical or random pores, is mapped into the globalpatient specific anatomic design for the ear or nose using Booleanintersection operations of the custom designed porous architecture .STLfile and the ear .STL file using MIMICS to create the final scaffolddesign.

The manufacturing process utilizes laser sintered polycaprolactone(PCL), an FDA approved, resorbable biopolymer. An EOS P100 lasersintering system is used with powder size, bed temperature, and lasersintering power as described in Partee B., et al., “Selective lasersintering process optimization for layered manufacturing of CAPA 6501Polycaprolactone Bone Tissue Engineering Scaffolds,” J. Manuf. Sci. E.128:531-540 (2006), the relevant portions of which are herebyincorporated by reference, to fabricate patient specific ear scaffolds.A 69% scaled male left ear is utilized. Random porosity is set at 61%.Spherical porosity is set at 65%. Pore size is 2.5 mm

In Vitro Cartilage Growth

Institutional Animal Care Committee protocol approval is obtained forthe study in this example. Chondrocytes are isolated from freshlyharvested porcine auricular cartilage. Care is taken to isolatecartilage while discarding overlying perichondrium. Minced cartilagefragments are digested with 0.2% type II collagenase (WorthingtonBiochemical, Lakeview, N.J.) for 16 hours in a 37° C., 5% CO₂ incubatorwith agitation. Digest is filtrated through a 70 micron mesh (BectonDickenson, Franklin Lakes, N.J.), the cells are centrifuged toprecipitate, then counted, and plated. The proliferation mediumcomprises of Ham's F-12 (Gibco, BRL/Life Technologies, Grand Island,N.Y.), with the addition of 10% fetal bovine serum (FBS, Sigma-Aldrich,St. Louis, Mo.), 5 mg/ml ascorbic acid, and an antibiotic/antimicoticsolution containing 10,000 U/ml penicillin, 10 mg/ml streptomycin, and25 μg/ml Fungizone.

Chondrocytes are seeded into the auricular PCL scaffolds using a type Icollagen/hyaluronic acid composite gel. The gel solution includes type 1collagen at a concentration of 6 mg/ml in acetic acid (Becton Dickinson,Frankin Lakes, N.J.) and hyaluronic acid at a concentration of 3 mg/ml(LifeCore Biomedical, Chaska, Minn.). Cells are rinsed with HanksBuffered Saline Solution (HBSS, Gibco, BRL/Life Technologies, GrandIsland, N.Y.), trypsinized (0.25% trypsin, Gibco), aliquoted into 15 mlconical tubes and placed on ice. Prior to seeding, the PCL scaffolds areplaced in custom-designed SYLGARD™ silicone (Dow Corning, Midland,Mich.) molds to prevent leakage of the cell-collagen solution prior togelation. After resuspending the cells in the collagen I gel solution,sodium bicarbonate is added, the cell suspension is carefully pipettedinto the PCL scaffolds and the constructs are placed in an incubator(37° C., 5% CO₂) for 30 minutes for gelation to occur. Approximately25×10⁶ chondrocytes are utilized per scaffold. Seeded constructs arecultured in sterile, dynamic conditions with incubation at 37° C., 5%CO₂. The culture medium includes serum free F12 (Gibco), with theaddition of 5 ng/ml TGF-β 2 (Pepro Tech, Rocky Hill, N.J.), ITS+premix(Becon Dickinson), 110 mm pyruvate (Gibco), 10 μm dexamethasone (Sigma),and 5 μg/ml ascorbic acid.

In Vivo Scaffold Implantation

NIH-Foxn1 strain 316, Charles River athymic rodents, 7-10 weeks of age,are implanted with seeded ear scaffolds after 4 weeks of in vitroculture. General anesthetic is administered. A dorsal incision isperformed with development of subcutaneous pocket. Layered skin closureis performed with 4-0 monocryl subcuticular closure.

After 9.5 weeks, ear constructs are histologically analyzed. Forhistology, 1 random and 1 spherical ear scaffold is divided in toquarters. The specimens are fixed with 10% phosphate buffered formalinfor 24 h, and then embedded in paraffin and sectioned using standardhistochemical techniques. Serial slide sections are stained withhematoxylin and eosin or Safranin O.

Auricular constructs with two micropore architectures, random andspherical, are rapidly manufactured with high fidelity anatomicappearance as shown in FIGS. 8A-8D. FIG. 8A shows a randomly distributedpore architecture, while FIG. 8B shows a regularly distributed patternof spherical pores in the tissue implant. FIG. 8C shows the PCL scaffoldplaced in a custom-designed SYLGARD™ silicone (Dow Corning, Midland,Mich.) mold to prevent leakage of a cell-collagen solution prior togelation. FIG. 8D shows the PCL scaffold after gelation of thecell-collagen solution on the surface.

Subcutaneous implantation of the scaffolds results in excellent externalappearance of both anterior and posterior auricular surfaces, as shownin FIGS. 9A-9C. Scaffold landmarks including helix, antihelix, conchalbowl, tragus, antitragus, and intertragal incisor are readily evidentafter subcutaneous implantation (FIGS. 9A-9B). Projection isapproximately 25-30° off horizontal plane of the animal dorsum (FIG.9C).

Histologic analysis displays more robust Safranin O staining for thespherical pore scaffolds in comparison to random pore scaffolds as shownin FIGS. 10A and 10B. FIG. 10A shows Safranin O staining for the randompore architecture scaffold, while FIG. 10B shows Safranin O staining forthe spherical pore structure. The posterior surface up on right dorsum,anterior surface up on left dorsum (left). Anterior face surface details(right upper) and anterior oblique view highlighting projection (rightlower). Cartilage growth is seen both in the peripheral and centralaspects of the auricular scaffolds. The cartilage growth is much greaterfor the spherical pore architecture, as shown in FIG. 10B. Growth isobserved adjacent to polycaprolactone structure though did not extendbeyond the confines of the scaffold as shown in FIGS. 10A-10B. H and Estaining additionally had more apparent cartilage matrix present in thespherical scaffolds.

This example further elucidates the potential benefit of various poredesigns. Previous work demonstrated the ability to direct pluripotentbone marrow derived stem cells toward either an osteocyte or chondrocytedifferentiation by adjusting pore design. Here, 3D printed ear scaffoldsare rapidly and consistently produced from bioresorbablepolycaprolactone. Though aesthetic appearance is not a primary goal ofthis example, the ear scaffolds reveal excellent detail whensubcutaneously implanted. Appearance is believed to be further improvedwith use of negative pressure vacuum suction.

Cartilage growth is observed in both scaffold designs in FIGS. 10A and10B, but appears to be more robust in the “chondrogenic” spherical poredesign. Furthermore, cartilage growth appears to be equally in theperipheral and central component of the scaffolds, though does notappear to grow beyond the boundaries of the scaffold. Therefore,creation of spherical micropores within the scaffold architectureappears to impart greater chondrogenicity of the scaffold, although bothtypes of pores promote cell growth in the scaffold.

Example B

This example explores a role of cellular population of 3D printed earscaffolds as compared to unseeded 3D printed scaffolds. This exampleestablishes the upper (resorbable 3D printed scaffold with seeded cells)and lower bounds (3D printed resorbable scaffold alone) of earreconstruction using tissue engineering. Without being limited to anyparticular theory, the theoretic optimal biologic scenario is believedto have the ear scaffold maximally seeded with a chondrocyte embeddedhydrogel. In this scenario, the goal of an ear scaffold is to becomeconfluent with ear cartilage. The theoretic optimal translationalscenario—by avoiding regulatory demands brought by using cell therapy—isutilizing an unseeded scaffold and inducing nearby cellular ingrowth. Itis believed that while both tissue scaffolds with predefined tetrahedralpores, seeded with chondrocyte embedding hydrogels and unseeded, willavoid distortion or contraction, the seeded scaffolds will exhibitimproved soft tissue coverage and have lower exposure and fracturerates.

Cell seeding of scaffold implants can be difficult to be able to harvestan adequate number of primary chondrocytes. Estimates of about 100 t toabout 150 million cells are believed to be likely needed to engineercartilage of human ear shape and volume. Preliminary data describedherein supports use of co-culture of primary chondrocytes with adiposederived stem cells (ADSC).

Microporous PCL scaffolds are seeded with porcine ADSCs and chondrocytesin experimental ratios of 1:1, 2:1, 5:1, and 10:1. Scaffolds are seededwith cells encapsulated in a hyaluronic acid/collagen hydrogel andcultured for 4 weeks without chondrogenic growth factors. Subcutaneousin vivo implantation of scaffolds is performed in an athymic rat model.NIH-Foxn1 strain 316, Charles River, 7-10 weeks of age rodent for ananimal model provides an excellent match to pediatric skin quality andthickness. Scaffolds are explanted after 4 weeks for histologic andbiochemical analysis. Histologic demonstration of cartilage growth isseen in all experimental groups. One-way ANOVA analysis demonstrated nosignificant difference in sulfated glycosaminoglycan (sGAG)/wet weight(ug/mg) concentration levels for 1:1, 2:1, and 5:1 experimental groups[F(2, 15)=0.028, α=0.05, p=0.97].

Immunohistochemistry demonstrates generous type II collagen depositionin all experimental groups is shown in FIG. 11 .

Both methods, seeding with chondrocytes alone or in co-culture withADSCs, meet the criteria for near term clinical translation. Bothmethods allow for autologous cell sources with harvest and seeding atthe time of scaffold implantation in the operating room.

Example C

This example is for determining the viability of utilizing a combinationof adipose-derived stem cell-chondrocyte co-culture andthree-dimensional (3D) printing to produce 3D bioscaffolds for cartilagetissue engineering. A feasibility study for cartilage tissue engineeringwith in vitro and in vivo animal data is described herein. Co-culture,where chondrocytes and mesenchymal stem cells (MSCs) are simultaneouslyseeded onto tissue engineering scaffolds, is used herein. Here the useof a co-culture model using adipose-derived stem cells (ASCs) andchondrocytes for cartilage tissue engineering (CTE) in conjunction withthe inventive ear implant scaffolds. The co-culture technique can beadapted for craniofacial cartilage applications using hydrogels combinedwith 3D-printed bioresorbable scaffolds and that a variety of ratios ofASCs-to-chondrocytes may be utilized. This approach affords thepotential for patient-specific CTE using computer-sided design (CAD)while mitigating the limitations of cell availability and need forprolonged in vitro cell culture or exogenous growth factor exposure oftraditional CTE approaches.

Protocol approval is obtained by the University of MichiganInstitutional Animal Care & Use Committee and the University of IllinoisInstitutional Animal Care & Use Committee (University of Michigan #3857,University of Illinois #10114).

Scaffold Design and Manufacturing Via 3D Printing

Scaffolds are created using previously described image-basedhierarchical design methods discussed above. This process can be used tocreate patient-specific tissue engineering scaffolds of any geometry. Astandard 10 mm×5 mm cylindrical disc scaffold macroarchitecture with a2.7 mm spherical pore internal microarchitecture is chosen for thisstudy to produce consistency of constructs for tissue analysis (FIG.2A). This yields an overall scaffold porosity of 68.3% with an availablevolume per scaffold of 268 μL. A midline groove is incorporated into thescaffold to facilitate bifurcation during analysis. The final scaffolddesign is then 3D printed using an EOS P100 laser sintering system (EOSNorth America, Novi, Mich.) adapted to laser sinter L-polycaprolactone(PCL) powder (PCL Source: Polysciences, Warrington, Pa.; PCLPreparation: Jet Pulverizer, Moorsetown, N.J.). The laser sinteringprocess can accurately reproduce feature sizes on the order of 70 μm andproduce over 500 representative scaffolds with a single print cycle.Scaffolds are cleaned of residual excess powder via sonication in 70%sterile ethanol then sterilized in a 24 hour 70% sterile ethanol soakprior to use.

Cell Harvest and Culture

Porcine ASCs derived from subcutaneous back fat and chondrocytes derivedfrom auricular and tracheal cartilage are harvested from adolescentYorkshire pigs using the methods previously developed by Wheeler andcolleagues. Primary (P0) ASC and chondrocyte cells are spun down andfrozen prior to cell seeding experiments. At the time of preparation forscaffold seeding, cells are thawed and expanded in growth mediacomprising of high-glucose Dulbecco's Modified Eagle's Medium (DMEM)(Gibco) with 10% fetal bovine serum (FBS) (Gibco), 1% pen/strep, and0.2% Fungizone in a 37° C., 5% CO₂ incubator. ASCs are expanded topassage 2 (P2) and chondrocytes expanded to passage 1 (P1) to providesufficient cells for seeding. Cells are passaged at 90% confluence.

Creation of Experimental Ratios and Scaffold Seeding

Porcine adipose-derived stem cells and chondrocytes are isolated andco-seeded at 1:1, 2:1, 5:1, 10:1, and 0:1 experimental ratios in ahyaluronic acid/collagen hydrogel in the pores of 3D-printedpolycaprolactone scaffolds to form 3D bioscaffolds for cartilage tissueengineering. Bioscaffolds are cultured in vitro without growth factorsfor 4 weeks then implanted into the subcutaneous tissue of athymic ratsfor an additional 4 weeks before sacrifice. Bioscaffolds are subjectedto histologic, immunohistochemical, and biochemical analysis.

More specifically, adipose-derived stem cells and chondrocytes arerinsed with Hank's buffered saline solution (HBSS) (Gibco), trypsinized(0.25% trypsin) (Gibco), and aliquoted into experimental ratios of 1:1,2:1, 5:1, 10:1, and 0:1 ASC-to-chondrocyte. Given that the cells areharvested from several animals, each cell type is pooled prior tocreation of experimental ratios. Each experimental group is thenre-suspended in a type I collagen:hyaluronic acid hydrogel solution andseeded into a prewet cylindrical PCL scaffold. The hydrogel comprises oftype I collagen at a concentration of 6 mg/mL in acetic acid (DiscoveryLabware) and hyaluronic acid at a concentration of 3 mg/mL (LifeCoreBiomedical). Prior to seeding, the PCL scaffolds are placed incustom-fabricated silicone (SYLGARD™ Dow Corning) molds to preventextravasation of the seeding solution prior to gelation. A 0.05N NaOH inNaCO₃ solution is used to induce gelation and scaffolds are subsequentlytransferred to 24-well low attachment plates (Fischer Scientific) forculture. The cell seeding density is 2×10⁶ cells/cm³ and total of 12scaffolds per experimental group are seeded.

In Vitro Culture

Seeded constructs are cultured in a sterile 37° C., 5% CO₂ incubatorwith agitation. Culture media comprises low-glucose DMEM with 10% FBS,1% pen/strep, and 0.2% Fungizone and is changed every 2-3 days. After 4weeks, six scaffolds from each experimental group are extracted forpost-in vitro biochemical analysis, while the remaining six are reservedfor in vivo implantation.

In Vivo Implantation

Seven athymic rats underwent implantation with tissue engineeringscaffolds under general anesthesia with isoflurane delivered by mask.All scaffolds are rinsed with HBSS prior to implantation. All animalsare male with each weighing between 250 and 305 g. Each animal isshaved, prepped with iodine solution after induction of anesthesia and avertical incision is sharply made on the dorsum of the animal A total of4 scaffolds per animal are implanted in a subcutaneous pocket intorandomized quadrants on the back of the animal. The incision is thenclosed with surgical staples, which are removed on post-operative dayseven. After 4 weeks, the animals are euthanized and the scaffolds areharvested for post-in vivo analysis.

Biochemical Analysis

Post-in vitro and post-in vivo specimens are split along the midlinegroove to double the number of constructs for analysis. One-half of eachconstruct is weighed wet, lyophilized, reweighed dry, and digested in 1mg/mL Papain stock solution (Fischer Scientific) at 65° C. for 16 hours.PicoGreen assay (Invitrogen, Colecular Probes) is used to quantify theDNA content of the constructs with Lambda phage DNA (0-1 mg/mL) as astandard. The sulfated-glycosaminoglycan (s-GAG) content is measuredusing the Blyscan Glycosaminoglycan Assay (Accurate Chemical &Scientific Corp).

Histology and Immunohistochemistry

Remaining post-in vivo constructs are fixed in 4% formalin for 24 hours,embedded in paraffin (TissuePrep, Fischer Scientific), and processedusing standard histologic procedures with a slice thickness of 10 μm.Stains included hematoxylin and eosin, Safrinin-O, and toluidine blue.Type II collagen immunohistochemical staining is performed using 5 μg/mLprimary mouse anti-type II collagen monoclonal antibodies (Hybridoma,University of Iowa).

Statistical Analysis

Data for biochemical analysis (DNA and s-GAG expression) are collectedfrom six samples after 4 weeks of in vitro cell culture and 4 weeks ofin vivo growth. Data is expressed as mean±standard error of the mean(SEM). Results are analyzed using Student's t-test using SPSS 17.0 (SPSSInc., Chicago, Ill.) and statistical significance is set to 5% (α=0.05)in all analyses.

Successful production of cartilage is achieved using a co-culture modelof adipose-derived stem cells and chondrocytes, without the use ofexogenous growth factors. Histology demonstrates cartilage growth forall experimental ratios at the post-in vivo time point confirmed withtype II collagen immunohistochemistry. There is no difference insulfated-glycosaminoglycan production between experimental groups.

Tissue engineered cartilage is successfully produced on 3D-printedbioresorbable scaffolds using an adipose-derived stem cell andchondrocyte co-culture technique. This potentiates co-culture as asolution for several key barriers to a clinically-translatable cartilagetissue engineering process.

Thus, in vitro co-culture of porcine ASCs and chondrocytes in 3D-printedPCL cylindrical discs with an internal spherical porous architectureresults in growth and maintenance of cartilage-like tissue after 8weeks. Surgical implantation is straightforward and the scaffolds arewell tolerated by the animals with no minor or major complications.There is good maintenance of structural support by the PCL scaffoldsafter 4 weeks growth in a subcutaneous pocket as shown in FIG. 2B.Histologically normal appearing cartilage growth is noted in allexperimental groups after 1 month of in vivo culture. The degree ofhistologic cartilage deposition is subjectively higher in theexperimental co-culture groups compared to the control group ofchondrocytes alone (FIG. 11 ), which is confirmed with type II collagenimmunohistochemistry. In particular, the 5:1 ASC-to-chondrocyte ratioprovided well delineated hyaline cartilage architecture in histology,with dense collagen deposition and lacunae surrounding the chrondrocytesand differentiated ASCs as shown in FIG. 12 .

Biochemical analysis results are summarized in FIGS. 13A-13B. There isno statistically significant difference in DNA/wet weight (ng/mg) ors-GAG/wet weight (μg/mg) content between the co-culture experimentalgroups at the post-in vitro or post-in vivo time points. There is astatistically significant higher s-GAG content in all co-culture groupscompared to the chondrocyte-alone control group (p<0.05 for allanalyses) at the post-in vitro timepoint, however this differencedisappeared at the post-in vivo timepoint.

Reconstruction of the auricular framework, whether performed in thesetting of trauma, oncologic resection, or congenital malformation, aresome of the most demanding procedures in facial reconstructive surgery.Tissue engineering holds several ubiquitous advantages, including theability to create a patient-specific living construct using thepatient's own cells. However, the primary limitation of utilizing solelychondrocytes for CTE is the large number of cells (up to 5×10⁷) neededto seed human-sized craniofacial frameworks. The number of chondrocytesavailable from autologous cartilage is limited and passagingchondrocytes induces dedifferentiation with loss of type II collagen andsulfated glycosaminoglycan (s-GAG) production. Mesenchymal stem cells,of which ample cell quantities are available, have been posited as asolution to seeding requirements. Prior experiments have shown a varietyof MSC types, including ASCs and bone-marrow stromal cells to be capableof chondrogenic differentiation. However, chondrogenic commitment ofMSCs requires exogenous delivery of pro-chondrogenic growth factors(GFs) for weeks and cells can demonstrate a propensity for ossification.

Co-culturing of chondrocytes and MSCs is the technique described in thisexample can circumvent the limitations of utilizing chrondrocytes orMSCs alone. In a co-culture model, chrondocytes and MSCs aresimultaneously seeded onto a tissue engineering scaffold. Chondrocyteshave been found to induce chondrogenic differentiation of the MSCs viaproduction of exogenous GFs such as cytokine-like protein 1 (Cytl1),bone morphogenic protein-2 (BMP-2), parathyroid hormone-related peptide(PTHrP), and transforming growth factor-beta (TGF-β) as well asparacrine, juxtacrine, and gap-junction signaling pathways. In this way,chondrocytes maintain the chondrogenic niche required for commitment ofMSCs to the chondrogenic phenotype, circumventing the need for exogenousGF delivery. Additionally, chondrocytes provide matrix for MSC migrationand prevent ossification of MSC-derived chondrocytes.

The process of forming an ear tissue scaffold implant according tocertain aspects of the present disclosure includes using CAD and 3Dprinting to produce high-fidelity patient-specific tissue engineeringscaffolds using PCL, a bioresorbable polymer. Utilization of abioresorbable material allows for eventual replacement of the scaffoldwith chondrocyte extracellular matrix, thus best emulating naturalcraniofacial cartilage. These scaffolds are then seeded with primarychondrocytes to produce tissue engineered auricular constructions. Thisprocess affords the ability to rapidly produce high-fidelity anatomicscaffolds while also allowing meticulous control of the poremicroarchitecture.

ASC co-culture on 3D-printed tissue engineering scaffolds for successfulCTE is believed to have been demonstrated for the first time. The use ofASCs with a co-culture technique is particularly advantageous for aclinically-translatable approach, given the low morbidity to harvestthese cells compared to bone-marrow derived stromal cells. The describedprocess can readily be adapted for tissue engineering constructs of anyshape, including patient-specific auricular and nasal constructs usingDICOM data

The results demonstrate that all experimental ratios ofASC-to-chondrocytes result in chondrocytic differentiation of ASCs onshort term in vivo analysis. Notably, chondrocytic commitment of theASCs is achieved without the use of exogenous GFs during scaffoldincubation. Using a cell count goal of 5×10⁷ as the number of cellsneeded for a typical human-sized auricle, ratios of 10:1 and 5:1 ASC:PCyield cell number requirements which are clinically achievable from acell harvest without the need to for prolonged passaging of cells in thelaboratory setting. This represents important barriers to aclinically-translatable process for craniofacial CTE which appear to beovercome with a co-culture technique.

Interestingly, the co-culture scaffold groups all appear to performsimilarly, despite different ratios of ASC-to-chondrocytes.Additionally, the co-culture groups appeared to outperform thechondrocyte-alone scaffolds, despite identical cell seeding densities.This may represent an inherent superior viability of co-cultured cellsin this methodology or synergistic interaction of co-cultured cells topromote chondrogenesis. However, this could also be an artifact ofdecreased viability of chondrocytes in cell culture. Given that noanalysis of cell viability or gene expression is performed in thisexample, these ideas are non-limiting and remain speculative.

This feasibility study is somewhat limited by a short in vivo incubationperiod and small number of implant constructs. As such, statisticaldifferences of the biochemical characteristics of the experimentalgroups may have not been captured, as well as differences in thetrajectory of tissue deposition with more prolonged in vivo growth.Given that the cells are seeded onto three-dimensional constructs, it isnot possible to perform cell viability or gene expression analysis.However, PCL constructs prepared in accordance with certain aspects ofthe present disclosure are believed to be able to maintain constructfidelity for 2 to 3 years prior to resorption.

The present example thus provides the successful use of anASC-chondrocyte co-culture technique and CAD-designed 3D-printed tissueengineering scaffolds for CTE in an animal model. This co-culture modelproduces formal cartilage production on short term in vivo follow-up inall experimental groups, including 5:1 and 10:1 ASC:chondrocyte ratios.The clinical availability of ASCs and lack of a need for prolongedexogenous GF exposure suggest this approach mitigates many of thelimitations of traditional CTE approaches. These represent key barriersto the eventual goal of creating a clinically translatablepatient-specific craniofacial CTE methodology that may be overcome usingco-culture and 3D printing.

In various aspects, multiple tissue scaffold ear implant embodiments aredescribed. The first is a single ear construct with the advantages of asingle operative procedure providing a final reconstructive outcome.Recognizing this may induce undesirable mechanical strain on vascularityin the overlying soft tissue, a second version implements amulticomponent tissue scaffold ear implant assembly. In certainembodiments, the implant assembly may include two distinct scaffoldcomponents that may have an interlock, such as a lock-in-key, dove taildesign. Such an implant assembly provides the benefits of the meticulousanatomic fidelity in the first stage implant, followed by a seamlesssecond stage elevation. This implant design is somewhat analogous to theNagata surgical technique of auricular reconstruction. While adding asecond operation, the potential benefit of a two stage implant is reliefof soft tissue vascular strain and minimization of framework extrusionand exposures. Finally, a novel “leaf insert” design that would allowfor calculated expansion of the framework in the second stage surgery,in concert with framework elevation, allows for the appearance offramework growth without necessitating cell therapy or growth factors.

The present disclosure thus contemplates methods that readily replicateany patient specific, complex auricular geometry, typically by mirroringthe unaffected contralateral auricle. In addition, the ear implants(e.g., PCL implant) have sufficient mechanical stiffness to resistcontraction and distortion. Importantly, the porous architecture can bedesigned to reduce the chance of dehiscence. The introduction of amodular design also provides the potential for early implantation inchildhood and expansion of the ear scaffold in a subsequent stagesurgery. A major limitation for implanting auricular devices in patientsyounger than 8 is matching growth of the contralateral ear. The abilityto print a modular expandable ear would allow earlier auricularreconstructions. Modular expansion is a novel feature for an earframework in ear reconstruction and is unique to the 3D printingprocesses. The scaffolds prepared in accordance with certain aspects ofthe present disclosure can potentiate a tissue engineering solutionwithout needing the addition of cells, while also having the possibilityto serve as a platform to deliver cells, tissue, and/or growth factorsto regenerate cartilaginous structures.

The foregoing description of the embodiments has been provided forpurposes of illustration and description. It is not intended to beexhaustive or to limit the disclosure. Individual elements or featuresof a particular embodiment are generally not limited to that particularembodiment, but, where applicable, are interchangeable and can be usedin a selected embodiment, even if not specifically shown or described.The same may also be varied in many ways. Such variations are not to beregarded as a departure from the disclosure, and all such modificationsare intended to be included within the scope of the disclosure.

What is claimed is:
 1. An implant for reconstruction of auricular tissuein a patient, the implant comprising: a tissue scaffold componentcomprising a biocompatible polymeric material having a plurality of openpores and that defines a portion of the patient's auricle of an ear,wherein the plurality of open pores includes at least one first openpore having a first diameter and a plurality of second open pores havinga second average diameter less than the first diameter, wherein the atleast one first open pore defines a hollow feature configured to receivea tissue sample, and the plurality of second open pores is distributedthroughout a body of the tissue scaffold component and is configured tosupport cell growth, and the tissue scaffold component has at least afirst region that is configured to define at least a portion of an outerear framework of the patient's auricle after implantation.
 2. Theimplant of claim 1, wherein the tissue sample is a punch biopsy insert.3. The implant of claim 1, wherein the tissue sample comprisescartilage.
 4. The implant of claim 1, wherein the at least one firstopen pore further comprises a plurality of first open pores disposedalong the first region of the tissue scaffold component that isconfigured to define at least the portion of the outer ear framework ofthe patient's auricle after implantation of the implant into thepatient.
 5. The implant of claim 1, wherein the plurality of open poresfurther comprises at least one third open pore having a third averagediameter that is distinct from the first diameter and greater than thesecond average diameter.
 6. The implant of claim 1, wherein thebiocompatible polymeric material is selected from the group consistingof: polycaprolactone (PCL), polyvinyl alcohol (PVA), polysebacic acid,polyethylene glycol (PEG), polylactic acid (PLA), polyethylene (PE),polyurethane (PU), extracellular tissue matrix, polysiloxane,polyetheretherketone (PEEK), polyetherketoneketone (PEKK), andcombinations thereof.
 7. The implant of claim 1, wherein the tissuescaffold component further comprises a bioactive agent selected from thegroup consisting of: a cell adhesion factor, a growth factor, a peptide,a cytokine, a hormone, a pharmaceutical active, and combinationsthereof.
 8. The implant of claim 1, wherein the tissue scaffoldcomponent further comprises a biomaterial selected from the groupconsisting of: an isolated tissue material, a hydrogel, acellularizeddermis, an acellularized tissue matrix, a composite of acellularizeddermis matrix and designed polymer, or a composite of acellularizedtissue matrix and polymer, and combinations thereof.
 9. The implant ofclaim 1, wherein the tissue scaffold component further comprises a drainport and an internal channel in fluid communication therewith.
 10. Theimplant of claim 1, wherein the first diameter of the at least one firstopen pore is greater than or equal to about 1 mm to less than or equalto about 12 mm and the first diameter is at least 100% greater than thesecond average diameter.
 11. The implant of claim 1, wherein the atleast one first open pore that defines the hollow feature furthercomprises a back stop rim or ledge configured to prevent displacement ofthe tissue sample during implantation of the implant into the patient.12. The implant of claim 12, wherein the hollow feature accepts aportion of the tissue sample and is configured to allow the tissuesample to protrude from the tissue scaffold component by greater than orequal to about 50 micrometers to less than or equal to about 10 mm. 13.An implant assembly for reconstruction of auricular tissue in a patient,the implant assembly comprising: a first tissue scaffold componentcomprising a first biocompatible polymeric material having a pluralityof open pores, wherein the plurality of open pores includes at least onefirst open pore having a first diameter and a plurality of second openpores having a second average diameter distinct from the first diameter,wherein the at least first one open pore defines a hollow featureconfigured to receive a tissue sample insert and the plurality of secondopen pores is distributed throughout a body of the first tissue scaffoldcomponent and is configured to support cell growth, wherein the firsttissue scaffold component defines a central void region and isconfigured to define at least a portion of an outer ear framework of thepatient after implantation; and a second tissue scaffold componentcomprising a second biocompatible polymeric material having a pluralityof third open pores configured to support cell growth, wherein thesecond tissue scaffold component defines a base portion and afterimplantation of the implant assembly into the patient, the second tissuescaffold component seats within the central void region of the firsttissue scaffold component, so that the second tissue scaffold componentis secured to the first tissue scaffold component, wherein the firstbiocompatible polymeric material and the second biocompatible materialmay be the same or distinct from one another.
 14. The implant assemblyof claim 13, wherein the first tissue scaffold component furthercomprises a first interlock member and the second tissue scaffoldcomponent further comprises a second interlock member, wherein afterimplantation into the patient, the first interlock member and the secondinterlock member are coupled together to secure the first tissuescaffold component to the second tissue scaffold component.
 15. Theimplant assembly of claim 14, wherein the first interlock member and thesecond interlock member together define a dove tail interlock assemblyor an offset snap assembly.
 16. The implant assembly of claim 13,wherein the second tissue scaffold component comprises a projection andthe first tissue scaffold component defines a void to receive theprojection, wherein the projection is configured to elevate the implantassembly.
 17. The implant assembly of claim 13, wherein the secondtissue scaffold component comprises a rectangular projection and thefirst tissue scaffold component defines a rectangular void to receivethe rectangular projection, wherein the rectangular projection isconfigured to elevate the implant assembly.
 18. The implant assembly ofclaim 13, wherein the at least one first open pore that defines thehollow feature further comprises a back stop rim or ledge configured toprevent displacement of the tissue sample during implantation of theimplant into the patient.
 19. The implant assembly of claim 18, whereinthe hollow feature accepts a portion of the tissue sample and isconfigured to allow the tissue sample to protrude from the first tissuescaffold component by greater than or equal to about 50 micrometers toless than or equal to about 10 mm.
 20. The implant assembly of claim 13,wherein the first biocompatible polymeric material and the secondbiocompatible polymeric material are independently selected from thegroup consisting of: polycaprolactone (PCL), polyvinyl alcohol (PVA),polysebacic acid, polyethylene glycol (PEG), polylactic acid (PLA),polyethylene (PE), polyurethane (PU), extracellular tissue matrix,polysiloxane, polyetheretherketone (PEEK), polyetherketoneketone (PEKK),and combinations thereof.